Preclinical studies on angiotensin converting enzyme inhibitors.

The identification of the renin-angiotensin-aldosterone system in the control of blood pressure, and the preclinical development of the angiotensin converting enzyme inhibitors for therapeutic use are reviewed. The properties of these compounds are discussed with respect to their in vitro enzyme inhibitory potency; prevention of the pharmacological effects of angiotensin I; potentiation of those of bradykinin; tissue enzyme inhibition; mechanism of effect on blood pressure both alone and in combination with other antihypertensive agents; and effect on cardiac parameters.

Biological properties of the angiotensin-converting enzyme inhibitor cilazapril.

Cilazapril is the monoethyl ester prodrug form of a potent, specific. long-acting antihypertensive inhibitor of angiotensin-converting enzyme (ACE). The biochemical and pharmacological properties of this compound have been compared with those of captopril and enalapril. In all test systems, cilazapril was the most potent and the longest acting. The active diacid of cilazapril was more potent than the corresponding diacid of enalapril in inhibiting the cleavage of angiotensin I and of Hip-His-Leu by ACE in vitro, in antagonising the angiotensin I-induced contractions of the isolated ileum of the guinea pig, in potentiating the vasodepressor responses to bradykinin, and in reducing the angiotensin I-induced rise in blood pressure of the rat. Parent drug absorption and diacid bioavailability in the rat were higher than for enalapril, and the inhibition of plasma ACE of longer duration. Single doses of cilazapril were more potent than enalapril in lowering the blood pressure of spontaneously hypertensive rats (SHR) and two-kidney renal hypertensive rats. On repeated daily oral dosing to SHR, both compounds had a cumulative antihypertensive effect. The acute antihypertensive effect was enhanced by simultaneous treatment with hydrochlorothiazide.

New potent inhibitors of angiotensin converting enzyme.

Using an earlier model of the favoured orientation of binding functions of angiotensin converting enzyme (ACE) inhibitors, it has been possible to postulate a new, 7,6-bicyclic system, based on hexahydropyridazine, which might be expected to have high potency. Some members of this system which have been synthesised have been shown to be very active ACE inhibitors, in vitro and in vivo.

Method for measuring the duration of inhibition of angiotensin I-converting enzyme in vivo.

The spontaneously hypertensive rat, either pithed or anesthetized with urethane, responds to intravenous injections of angiotensin I (A1) and angiotensin II (AII) with pressor responses. These responses vary with time, but the ratio of the responses to AI and AII is constant throughout 4.5 hr. This criterion was used to determine the degree and the duration of effect of captopril. At low (30 micro g/kg iv) and high (3mg/kg iv) doses, it selectively reduces the pressor response to AI, so reducing the AI/AII ratio. The rate of recovery is initially rapid followed by a prolonged slower phase. The degree (maximum 65%) and the duration of inhibition are directly related to the dose of the inhibitor.

A method for the differentiation of presynaptic inhibitors of adrenergic neurotransmitter release.

Injection of clonidine hydrochloride, 30 microgram/kg iv, or bretylium tosylate, 5 mg/kg iv, in pithed male spontaneously hypertensive rats inhibits the pressor responses to stimulation of the total sympathetic outflow, and enhances those to injected noradrenaline. The intravenous injection of d-amphetamine sulphate, 200 microgram/kg, reverses bretylium inhibition of the responses to sympathetic stimulation but not that of clonidine. Yohimbine, 10 microgram/kg/min iv on the other hand, reverses clonidine inhibition of the responses to sympathetic stimulation, but not that of bretylium. This pharmacological analysis provides a method for the differentiation of the mechanism of effect of two types of antihypertensive drug which act presynaptically to impair the release of the neurotransmitter from sympathetic nerves, viz., sympathetic neurone blocking agents (such as bretylium tosylate) and presynaptic alpha-adrenoceptor stimulants (such as clonidine hydrochloride).

The in vivo examination of the irreversible beta-adrenoceptor antagonist Ro 03-7894 on cardiac rate and contractility.

1. Two benzofuran-2-ethanolamines Ro 03-5255 (1-(5-acetylamino-benzofuran-2-yl)-1-hydroxy-2-isopropylaminoethane) and Ro 03-7894 (1-(5-chloracetyl aminobenzofuran-2-yl)-1-hydroxy-2-isopropylaminoethane) which had previously been shown to exhibit respectively competitive and irreversible beta-adrenoceptor antagonism in guinea-pig isolated atria, were compared in vivo using isoprenaline-induced tachycardia of anaesthetized guinea-pigs and heart rate and contractility (dp/dtmax) of open-chest anaesthetized guinea-pigs and of conscious cats. 2. In urethane-anaesthetized guinea-pigs doses of 3 mg/kg, s.c. of both antagonists produced significant blockade of the rate response to an 80% of maximum dose of isoprenaline after 4 h. In other experiments, guinea-pigs were pretreated with the antagonists and the responses to isoprenaline were then monitored. The slopes of the dose-response curves to isoprenaline were depressed for up to 24 h by Ro 03-7894 but this was not so with Ro 03-5255. 3. In conscious cats the course of blockade by Ro 03-7894 was followed in the same animals and was still evident after 48 h. In contrast, the beta-adrenoceptor blockage produced by Ro 03-5255 was not evident 24 h after administration. 4. The persistence of blockade by Ro 03-7894 was consistent with the irreversible mode of action demonstrated in vitro.

Quantitative studies of the effect of antagonists on the acute toxicity of organophosphates in rats.

1. The subcutaneous acute toxicities of the vinyl phosphate pesticides monocrotophos, dicrotophos, chlorfenvinphos, crotoxyphos, dichlorvos, mevinphos, and of the experimental compounds SD 4455 (cis-2-carboxy-1-methylvinyl dimethylphosphate) and SD 7779 (cis-2-(1-phenylethoxy) carbonyl-1-methylvinyl diethylphosphate) have been determined in female rats.2. The effects on the log dose-probit mortality curves to the vinylphosphates of the therapeutic subcutaneous administration of methylatropine, atropine, N-methylpyridinium-2-aldoxime methanesulphonate and obidoxime have been studied.3. Elevation of the LD50 values by the therapeutic regimens was shown to be an unsatisfactory measure of therapeutic efficiency, while reduction of the effect of a maximally lethal dose (LD90) to less than that of a minimally lethal dose (LD10) provided a better quantitative measure of therapeutic efficiency.4. The combination of atropine sulphate (50 mumol base/kg) with obidoxime (250 mumol/kg) was found to be generally the most effective of the antidotal regimens.

The pharmacology of the cholinoceptor in muscle preparations of Ascaris lumbricoides var. suum.

1. The preparation of a muscle strip of Ascaris lumbricoides for the study of the effect of drugs in vitro is described.2. Stimulant drugs which are classified as nicotine-like in mammalian pharmacology increased the isometric tension of this preparation. These drugs were, in descending order of potency: dimethylphenylpiperazinium, nicotine, acetylcholine, carbachol, decamethonium and pyridine-2-aldoxime methiodide.3. Muscarine-like drugs (oxotremorine, methacholine, pilocarpine) had no activity.4. Potassium and barium ions stimulated the tissue, while the anti-cholinesterases, dichlorvos and eserine, increased the resting tension of the preparation and potentiated the responses to acetylcholine.5. Adrenaline neither stimulated the tissue nor affected the responses to nicotine-like drugs.6. The relative potency of several blocking agents which antagonize the responses to nicotine-like drugs was assayed. These blocking agents were, in descending order of potency: mecamylamine, (+)-tubocurarine, hexamethonium, atropine and piperazine. Acetylcholine, dimethylphenylpiperazinium and pyridine-2-aldoxime methiodide apparently act on a common receptor, for each blocking agent had a similar molar inhibitory concentration against these stimulants.7. It is concluded that the cholinoceptor in muscle preparations of Ascaris lumbricoides is pharmacologically similar to that of the mammalian autonomic ganglion.